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Angiogenesis plays an important role in the growth and metastasis of solid tumors including melanoma. Inhibiting tumor-associated angiogenesis is a tactic in treating melanoma. Dioscin restrains angiogenesis in colon tumor and has anti-melanoma effects in cell and animal models. In a previous study, we found that dioscin inhibits Src/STAT3 signaling in melanoma cells. Activation of the Src/STAT3 pathway has been shown to promote tumor angiogenesis. This study aimed to determine whether dioscin's anti-melanoma effects is related to inhibiting Src/STAT3 signaling-mediated angiogenesis. In a B16F10 allograft mouse model, we found that dioscin inhibited melanoma growth and angiogenesis. To exclude the impact of tumor growth on angiogenesis, a chicken chorioallantoic membrane (CAM) model was used to verify the anti-angiogenic effect of dioscin. Results showed that dioscin suppressed vessel formation in CAM. To determine if tumor secreted pro-angiogenic cytokines are involved in the anti-angiogenic effect of dioscin, conditioned media from dioscin-treated A375 melanoma cells were used to culture human umbilical vein endothelial cells (HUVECs), and tube formation was monitored. It was observed that the tube formation of HUVECs was inhibited. Mechanistic studies revealed that dioscin inhibited the activation of Src and STAT3, and lowered mRNA and protein levels of STAT3 transcriptionally-regulated genes, in B16F10 melanomas. ELISA assays showed that dioscin decreased the secretion of MMP-2, MMP-9 and VEGF from A375 cells. Over-activation of STAT3 lessened the effects of dioscin in decreasing the secretion of pro-angiogenic cytokines from melanoma cells, and in inhibiting tube formation of HUVECs cultured with conditioned media from melanoma cell cultures. In summary, we for the first time demonstrated that inhibiting Src/STAT3 signaling-mediated angiogenesis is involved in the anti-melanoma effects of dioscin. This study provides further pharmacological groundwork for developing dioscin as an anti-melanoma agent.
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Inibidores da Angiogênese/uso terapêutico , Diosgenina/análogos & derivados , Melanoma Experimental/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Fator de Transcrição STAT3/antagonistas & inibidores , Quinases da Família src/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Animais , Linhagem Celular Tumoral , Diosgenina/farmacologia , Diosgenina/uso terapêutico , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Fator de Transcrição STAT3/metabolismo , Carga Tumoral/efeitos dos fármacos , Quinases da Família src/metabolismoRESUMO
Bronchopulmonary dysplasia (BPD) is a chronic lung disease due to impaired pulmonary development and is one of the main causes of respiratory failure in preterm infants. Preterm infants with BPD have significantly higher complication and mortality rates than those without BPD. At present, comprehensive management is the main intervention method for BPD, including reasonable respiratory and circulatory support, appropriate enteral nutrition and parenteral nutrition, application of caffeine/glucocorticoids/surfactants, and out-of-hospital management after discharge. The continuous advances in stem cell medicine in recent years provide new ideas for the treatment of BPD. Various pre-clinical trials have confirmed that stem cell therapy can effectively prevent lung injury and promote lung growth and damage repair. This article performs a comprehensive analysis of the mechanism of mesenchymal stem cells in the treatment of BPD, so as to provide a basis for clinical applications.
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Displasia Broncopulmonar , Células-Tronco Mesenquimais , Displasia Broncopulmonar/prevenção & controle , Nutrição Enteral , Humanos , Recém-Nascido , Recém-Nascido Prematuro , PulmãoRESUMO
Primary liver cancer is a lethal cancer. The phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway has been implicated in the pathogenesis of liver cancer. Gomisin N (GN), a lignan isolated from the dried fruits of Schisandra chinensis (Turca.) Baill., has been reported to reduce viability of, and induce apoptosis in, HepG2 liver cancer cells. In preadipocytes, GN was found to inhibit Akt activity. In the present study, Akt signaling-related anti-liver cancer mechanisms of GN were investigated. We confirmed that GN reduces cell viability of, and triggers apoptosis in, more liver cancer cell lines. Mechanistic studies revealed that GN lowers protein levels of phospho-PI3K (p85 tyrosine (Tyr)458), phospho-Akt (serine (Ser)473), and Akt downstream molecules Mcl-1 in HepG2 and HCCLM3 cells. Meanwhile, GN activates mTOR and inhibits ULK1 (a negative downstream effector of mTOR) activities. Activation of mTOR has been reported to suppress ULK1 activity and repress autophagy. Indeed, we observed that GN inhibits autophagy in liver cancer cells. In summary, we for the first time demonstrated that GN inhibits the PI3K-Akt pathway and regulates the mTOR-ULK1 pathway in liver cancer cells.
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Antineoplásicos/farmacologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Lignanas/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Fosfatidilinositol 3-Quinase/fisiologia , Compostos Policíclicos/farmacologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Linhagem Celular Tumoral , Ciclo-Octanos/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
A traditional Chinese medicine (TCM) formula (SL) comprising Sophorae Flos and Lonicerae Japonicae Flos was used for treating melanoma in ancient China. We have previously shown that an ethanolic extract of SL (SLE) possesses anti-melanoma effects and suppresses STAT3 signaling in vitro and in vivo. STAT3 has been linked to the development of melanoma immunosuppressive microenvironment. In this work, we investigated whether SLE inhibits melanoma growth by reprogramming the tumor microenvironment in mouse and co-culture cell models. In B16F10 melanoma-bearing mice, we found that intragastric administration of SLE (1.2 g/kg) dramatically inhibited tumor growth. This observation was associated with the downregulation of protein levels of phospho-STAT3 (Tyr 705) and STAT3-regulated immunosuppressive cytokines, and mRNA levels of STAT3-targeted genes involved in tumor growth and immune evasion. We also observed increased Th, Tc and dendritic cells in the melanomas and spleens in SLE-treated mice compared to that in control mice. In a co-culture system composed of B16F10 cells and mouse primary splenic lymphocytes, it was found that SLE not only inhibited STAT3 activation in B16F10 cells, but also downregulated mRNA levels of STAT3-targeted genes in the splenic lymphocytes. In this co-culture setting, SLE decreased the levels of STAT3-regulated immunosuppressive cytokines, increased the percentages of Th, Tc and dendritic cells as well. Furthermore, effects of SLE on STAT3 phosphorylation, cytokine levels and immune cell subtype percentages were significantly weaker in the B16STAT3C cells (stable cells harboring a constitutively active STAT3 variant STAT3C)/splenic lymphocytes co-culture system than in the B16V cells (cells stably transfected with the empty vector)/splenic lymphocytes co-culture system, indicating that STAT3 over-activation diminishes SLE's effects. In summary, our findings indicate that reprograming the immune microenvironment, partially mediated by inhibiting STAT3 signaling, contributes to the anti-melanoma mechanisms of SLE. This study provides further pharmacological groundwork for developing SLE as a modern agent for melanoma prevention/treatment, and supports the notion that reprograming immunosuppressive microenvironment is a viable anti-melanoma strategy.
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Antineoplásicos/farmacologia , Melanoma Experimental/imunologia , Extratos Vegetais/farmacologia , Fator de Transcrição STAT3/imunologia , Neoplasias Cutâneas/imunologia , Sophora , Microambiente Tumoral/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Técnicas de Cocultura , Flores , Lonicera , Linfócitos , Masculino , Medicina Tradicional Chinesa , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/imunologia , Neovascularização Patológica/patologia , Extratos Vegetais/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Microambiente Tumoral/imunologiaRESUMO
Purpose: To examine the mediating effect of attitudes towards dementia on the relationship between dementia knowledge and behaviors towards persons with dementia. Participants and Methods: A cross-sectional survey was conducted with 313 adults (age ≤ 20 years). Participants were recruited using non-probability convenience sampling from medical clinics, community centers, and supermarkets located in the Wanhua District of Taipei City. Data were collected with the following self-report questionnaires: a demographic survey, validated instruments for dementia knowledge and attitudes towards dementia (assessed using the Alzheimer's Disease Knowledge Scale and the Approaches to Dementia Questionnaire, respectively), and a researcher-developed survey on unfriendly behaviors towards persons with dementia. Results: Pearson's correlation and multiple linear regression analysis indicated that higher scores for dementia knowledge and more positive attitudes about dementia were significantly associated with lower levels of unfriendly behaviors towards persons living with dementia. Mediation analysis using a robust bootstrap test with 5000 samples indicated that attitudes toward dementia had a partial mediating effect on the relationship between dementia knowledge and unfriendly behaviors. Conclusion: Our findings suggest that increasing public awareness and knowledge about dementia could help the general population develop better attitudes towards dementia, which could subsequently help improve behaviors towards persons living with dementia.
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BACKGROUND: A tri-herb formulation comprising Ganoderma (the dried fruiting body of Ganoderma lucidum), Puerariae Thomsonii Radix (the dried root of Pueraria thomsonii) and Hoveniae Semen (the dried mature seed of Hovenia acerba) -GPH for short- has been using for treating liver injury; however, the pharmacological basis of this application of GPH is unknown. This study aimed to investigate the liver protective effects and mechanisms of action of an ethanolic extract of GPH (GPHE) in mice. METHODS: To control the quality of GPHE, the contents of ganodermanontriol, puerarin and kaempferol in the extract were quantified by ultra-performance liquid chromatography. An ethanol (6 ml/kg, i.g.)-induced liver injury ICR mouse model was employed to investigate the hepatoprotective effects of GPHE. RNA-sequencing analysis and bioassays were performed to reveal the mechanisms of action of GPHE. RESULTS: The contents of ganodermanontriol, puerarin and kaempferol in GPHE were 0.0632%, 3.627% and 0.0149%, respectively. Daily i.g. administration of 0.25, 0.5 or 1 g/kg of GPHE for 15 consecutive days suppressed ethanol (6 ml/kg, i.g., at day 15)-induced upregulation of serum AST and ALT levels and improved histological conditions in mouse livers, indicating that GPHE protects mice from ethanol-induced liver injury. Mechanistically, GPHE downregulated the mRNA level of Dusp1 (encoding MKP1 protein, an inhibitor of the mitogen-activated protein kinases JNK, p38 and ERK), and upregulated expression and phosphorylation of JNK, p38 and ERK, which are involved in cell survival in mouse liver tissues. Also, GPHE increased PCNA (a cell proliferation marker) expression and reduced TUNEL-positive (apoptotic) cells in mouse livers. CONCLUSION: GPHE protects against ethanol-induced liver injury, and this effect of GPHE is associated with regulation of the MKP1/MAPK pathway. This study provides pharmacological justifications for the use of GPH in treating liver injury, and suggests that GPHE has potential to be developed into a modern medication for managing liver injury.
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Doença Hepática Crônica Induzida por Substâncias e Drogas , Etanol , Camundongos , Animais , Etanol/farmacologia , Quempferóis/farmacologia , Doença Hepática Crônica Induzida por Substâncias e Drogas/patologia , Camundongos Endogâmicos ICR , Fígado , Fosfatases da Proteína Quinase Ativada por Mitógeno/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
BACKGROUND: Pancreatic cancer has been characterized by poor prognosis, early metastasis and dissatisfactory treatment outcome. The high basal level of autophagy in tumor cells leads to chemoresistance and tumor progression. Thus, it is imminent to explore novel effective chemotherapeutic adjuvants to increase patients' survival rate. Isoliquiritigenin (ISL) is a bioactive flavonoid obtained from the Traditional Chinese herbal medicine Glycyrrhiza glabra, and it possesses a broad range of pharmacological effects. In this study, the anti-cancer effect of ISL in pancreatic cancer treatment and the underlying mechanism are investigated. METHODS: MTT assay, colony formation and EdU analysis were performed to explore the growth inhibition of ISL on pancreatic cancer cells. Apoptosis were analyzed using TUNEL and flow cytometry. The formations of autophagosomes were analyzed by immunofluorescence microscopy and transmission electron microscopy. RFP-GFP-LC3B probe was applied to detect the autophagy flux. To assess the structural interaction of ISL with p38 protein, molecular docking assays were performed. The molecular mechanism was elucidated by using western immunoblotting. Subsequently, the inhibition of ISL on tumor growth was determined in vivo using pancreatic tumor mice model. RESULTS: ISL inhibited pancreatic cancer cell growth and induced apoptosis, both in vitro and in vivo. ISL caused accumulation of autophagosome through blockade of late stage autophagic flux. Moreover, autophagy inducer rapamycin enhanced ISL-evoked cell growth inhibition and promoted apoptosis, while inhibition of autophagosome formation by siAtg5 attenuated ISL-induced apoptosis. It is remarkable that ISL synergistically sensitized the cytotoxic effect of gemcitabine and 5-fluorouracil on pancreatic cancer cells as both drugs induced autophagy. Molecular docking analysis has indicated that ISL acted by direct targeting of p38 MAPK, which was confirmed by ISL-induced phosphorylation of p38. The autophagy flux induced by p38 inhibitor SB203580 was blocked by ISL, with further increasing toxicity of ISL in pancreatic cancer cells. CONCLUSION: The results have revealed that ISL inhibited pancreatic cancer progression by blockade of autophagy through p38 MAPK signaling.
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Chalconas , Medicamentos de Ervas Chinesas , Neoplasias Pancreáticas , Animais , Apoptose , Autofagia , Linhagem Celular Tumoral , Chalconas/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Fluoruracila/farmacologia , Camundongos , Simulação de Acoplamento Molecular , Neoplasias Pancreáticas/tratamento farmacológico , Sirolimo/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Pancreatic cancer is amongst the most lethal malignancies, while its poor prognosis could be associated with promotion of autophagy and the tumor immune microenvironment. Studies have confirmed the pro-tumorigenic nature of the cathelicidin family of peptide LL-37 in several types of cancer. However, at higher doses, LL-37 exerts significant cytotoxicity against gastrointestinal cancer cells. In our study, we investigated the anti-tumorigenic potential of LL-37 in pancreatic cancer and the underlying mechanisms. Our results have shown that LL-37 inhibited the growth of pancreatic cancer both in vitro and in vivo. Mechanistic studies have demonstrated that LL-37 induced DNA damage and cell cycle arrest through induction of reactive oxygen species (ROS). Further study indicates that LL-37 suppressed autophagy in pancreatic cancer cells through activation of mTOR signaling, leading to more accumulation of ROS production and induction of mitochondrial dysfunctions. With combined treatment of LL-37 with the mTOR inhibitor rapamycin, LL-37-induced ROS production and cancer cell growth inhibition were attenuated. Subsequent in vivo study has shown that LL-37 downregulated the immunosuppressive myeloid-derived suppressor cells and M2 macrophages while upregulated the anti-cancer effectors CD8+ and CD4+ T cells in the tumor microenvironment. By using an in vitro co-culture system, it was shown that promotion of M2 macrophage polarization would be suppressed by LL-37 with inhibition of autophagy, which possessed significant negative impact on cancer growth. Taken together, our findings implicate that LL-37 could attenuate the development of pancreatic cancer by suppressing autophagy and reprogramming of the tumor immune microenvironment.
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BACKGROUND: Fibroblast-like synoviocytes (FLS) have cancer cell-like characteristics, such as abnormal proliferation and resistance to apoptosis, and play a pathogenic role in rheumatoid arthritis (RA). Hyperproliferation of RA-FLS that can be triggered by the activation of interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling destructs cartilage and bone in RA patients. Chrysoeriol is a flavone found in medicinal herbs such as Chrysanthemi Indici Flos (the dried capitulum of Chrysanthemum indicum L.). These herbs are commonly used in treating RA. Chrysoeriol has been shown to exert anti-inflammatory effects and inhibit STAT3 signaling in our previous studies. This study aimed to determine whether chrysoeriol inhibits hyperproliferation of RA-FLS, and whether inhibiting STAT3 signaling is one of the underlying mechanisms. METHODS: IL-6/soluble IL-6 receptor (IL-6/sIL-6R)-stimulated RA-FLS were used to evaluate the effects of chrysoeriol. CCK-8 assay and crystal violet staining were used to examine cell proliferation. Annexin V-FITC/PI double staining was used to detect cell apoptosis. Western blotting was employed to determine protein levels. RESULTS: Chrysoeriol suppressed hyperproliferation of, and evoked apoptosis in, IL-6/sIL-6R-stimulated RA-FLS. The apoptotic effect of chrysoeriol was verified by its ability to cleave caspase-3 and caspase-9. Mechanistic studies revealed that chrysoeriol inhibited activation/phosphorylation of Janus kinase 2 (JAK2, Tyr1007/1008) and STAT3 (Tyr705); decreased STAT3 nuclear level and down-regulated protein levels of Bcl-2 and Mcl-1 that are transcriptionally regulated by STAT3. Over-activation of STAT3 significantly diminished anti-proliferative effects of chrysoeriol in IL-6/sIL-6R-stimulated RA-FLS. CONCLUSIONS: We for the first time demonstrated that chrysoeriol suppresses hyperproliferation of RA-FLS, and suppression of JAK2/STAT3 signaling contributes to the underlying mechanisms. This study provides pharmacological and chemical justifications for the traditional use of chrysoeriol-containing herbs in treating RA, and provides a pharmacological basis for developing chrysoeriol into a novel anti-RA agent.
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Artrite Reumatoide , Flavonas , Sinoviócitos , Artrite Reumatoide/tratamento farmacológico , Fibroblastos , Flavonas/farmacologia , Humanos , Janus Quinase 2/metabolismo , Fator de Transcrição STAT3/metabolismo , Sinoviócitos/metabolismo , Sinoviócitos/patologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Malignant melanoma is a fatal cancer. Signal transducer and activator of transcription 3 (STAT3) has been proposed as a therapeutic target of melanoma. An herbal formula Huai-Hua-San (HHS) comprising Sophorae Flos (SF) and Gardeniae Fructus (GF) is traditionally used for treating cancers including melanoma, but the pharmacological basis is unknown. AIMS OF THIS STUDY: This study aimed to investigate the anti-melanoma effects of an ethanolic extract of HHS (HHSE), and explore the involvement of STAT3 signaling in the effects. MATERIALS AND METHODS: An UPLC-TOF/MS method was developed to control the quality of HHSE. A B16F10 allograft mouse model and three melanoma cell lines (B16F10, A375 and A2058) were used to determine the anti-melanoma effects of HHSE. Dacarbazine (DTIC) and Stattic were used as positive controls. Cell viability was detected using MTT and crystal violet staining assays. Cell apoptosis was analyzed by flow cytometry after the cells were stained with Annexin-V/PI. Cell invasive ability was examined using the transwell assay. Protein levels were determined by Western blotting. RESULTS: The contents of crocin I, crocin II, quercetin and kaempferol in HHSE were 0.59%, 0.98%, 4.66% and 1.15%, respectively. A clinically relevant dose of HHSE (0.1 g/kg/day, i.g. for 15 consecutive days) significantly suppressed B16F10 tumor growth in mice. HHSE dose-dependently reduced cell viability and dampened invasion of, and induced apoptosis in, melanoma cells. Mechanistic studies revealed that HHSE inhibited the phosphorylation/activation of STAT3 in B16F10 allografts and in cultured melanoma cells. In cell models, HHSE also inhibited the phosphorylation of STAT3 upstream kinases, JAK2 (Tyr1007/1008) and Src (Tyr416), lowered STAT3 nuclear levels, and down-regulated the protein levels of STAT3-targeted molecules. Over-activation of STAT3 in A375 cells significantly attenuated the cytotoxic effects of HHSE. CONCLUSIONS: HHSE exhibits anti-melanoma effects in cell and mouse models. Inhibition of STAT3 signaling contributes to the anti-melanoma mechanisms of HHSE. Our findings lay a groundwork for developing HHSE as a modern agent for melanoma management, and provide pharmacological justifications for the traditional use of HHS in treating melanoma.
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Antineoplásicos Fitogênicos/uso terapêutico , Melanoma Experimental/tratamento farmacológico , Preparações de Plantas/uso terapêutico , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos Fitogênicos/farmacologia , Relação Dose-Resposta a Droga , Humanos , Masculino , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Preparações de Plantas/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Fibroblast-like synoviocytes (FLS) play a pathogenic role in rheumatoid arthritis (RA). STAT3 signaling is activated in FLS of RA patients (RA-FLS), which in turn causes RA-FLS hyperproliferation. RL is a traditional remedy for treating inflammatory diseases in China. It comprises Rosae Multiflorae Fructus and Lonicerae Japonicae Flos. A standardized ethanolic extract of RL (RLE) has been shown to exert anti-arthritic effects in collagen-induced arthritis (CIA) rats. Some constituents of RLE were reported to inhibit JAK2/STAT3 signaling in rat FLS. Here, we determined whether RLE inhibits FLS hyperproliferation, and explored the involvement of STAT3 signaling in this inhibition. In joints of CIA rats, RLE increased apoptotic FLS. In IL-6/sIL-6R-stimulated RA-FLS, RLE reduced cell viability and evoked cell apoptosis. In synovial tissues of CIA rats, RLE lowered the protein level of phospho-STAT3. In IL-6/sIL-6R-stimulated RA-FLS, RLE inhibited activation/phosphorylation of STAT3 and JAK2, decreased the nuclear localization of STAT3, and downregulated protein levels of Bcl-2 and Mcl-1. Over-activation of STAT3 diminished RLE's anti-proliferative effects in IL-6/sIL-6R-stimulated RA-FLS. In summary, RLE inhibits hyperproliferation of FLS in rat and cell models, and suppression of STAT3 signaling contributes to the underlying mechanisms. This study provides further pharmacological groundwork for developing RLE as a modern anti-arthritic drug.
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Artrite Reumatoide/tratamento farmacológico , Medicamentos de Ervas Chinesas/uso terapêutico , Extratos Vegetais/uso terapêutico , Rosa , Sinoviócitos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Artrite Reumatoide/metabolismo , Avaliação Pré-Clínica de Medicamentos , Medicamentos de Ervas Chinesas/farmacologia , Humanos , Interleucina-6 , Lonicera , Fitoterapia , Cultura Primária de Células , Ratos , Fator de Transcrição STAT3/metabolismo , Líquido Sinovial/metabolismoRESUMO
Malignant melanoma is aggressive and has a high mortality rate. Toll-like receptor 4 (TLR4) has been linked to melanoma growth, angiogenesis and metastasis. However, signal transduction mediated by TLR4 for driving melanoma progression is not fully understood. Signal transducer and activator of transcription 3 (STAT3) has been identified as a major oncogene in melanoma progression. We found: that TLR4 expression positively correlates with activation/phosphorylation of STAT3 in human melanoma samples; that TLR4 ligands activate STAT3 through MYD88 and TRIF in melanoma cells; and that intratumoral activation of TLR4 increases STAT3 activation in the tumor and promotes tumor growth, angiogenesis, epithelial-mesenchymal transition (EMT) and the formation of an immunosuppressive tumor microenvironment in mice. Further, we found that the effects mediated by activating TLR4 are weakened by suppressing STAT3 function with a dominant negative STAT3 variant in melanoma. Collectively, our work identifies STAT3 activation as a key event in TLR4 signaling-mediated melanoma progression, shedding new light on the pathophysiology of melanoma.
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Melanoma/tratamento farmacológico , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Lipopolissacarídeos/farmacologia , Melanoma/metabolismo , Neovascularização Patológica/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
Late stage melanoma is associated with a high mortality rate. Signal transducer and activator of transcription 3 (STAT3) is currently a target for melanoma treatment as it is constitutively activated with high frequency in melanoma. Dioscin is a natural steroid saponin that is present in several medical herbs. A previous study demonstrated that dioscin inhibits STAT3 signaling in a cerebral ischemia-reperfusion injury rat model. Furthermore, dioscin has been reported to exert anti-melanoma effects in B16 melanoma cells and a B16 allograft mouse model. The present study investigated whether inhibition of STAT3 signaling is involved in the anti-melanoma effects of dioscin. The results of the present study demonstrated that dioscin significantly decreased viability, induced apoptosis and suppressed migration of human A375 melanoma cells and murine B16F10 melanoma cells. Furthermore, dioscin inhibited the phosphorylation of STAT3 and Src (an upstream kinase of STAT3), and downregulated mRNA levels of STAT3-targeted genes, including B-cell lymphoma-2, cyclin D1 and matrix metalloproteinase-2. In addition, overexpression of STAT3 decreased the anti-proliferative effects of dioscin. Overall, the results of the present study indicate that inhibiting the Src/STAT3 signaling pathway contributes to the anti-melanoma molecular mechanisms of dioscin. These results provide further pharmacological groundwork for developing dioscin as a novel anti-melanoma agent.
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BACKGROUND: Chrysoeriol is a flavone found in diverse dietary and medicinal herbs such as Lonicerae Japonicae Flos (the dried flower bud or newly bloomed flower of Lonicera japonica Thunb.). These herbs are commonly used for treating inflammatory diseases. Herbal extracts containing chrysoeriol have been shown to have anti-inflammatory effects and inhibit nuclear factor-kappa B (NF-κB) signaling. Some of these extracts can inhibit signal transducers and activators of transcription 3 (STAT3) signaling in cancer cells. PURPOSE: This study aimed to determine whether chrysoeriol has anti-inflammatory effects and whether NF-κB and STAT3 pathways are involved in the effects. STUDY DESIGN AND METHODS: A TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ear edema mouse model and LPS-stimulated RAW264.7 cells were used to evaluate the effects of chrysoeriol. Griess reagent was used to measure the production of nitric oxide (NO). Western blot and enzyme-linked immunosorbent assays were employed to detect protein levels. RT-qPCR analyses were used to detect mRNA levels. Haematoxylin and eosin (H&E) staining was employed to examine the pathological conditions in animal tissues. RESULTS: In the mouse model, chrysoeriol ameliorated acute skin inflammation, evidenced by reduced ear thickness, ear weight and number of inflammatory cells in inflamed ear tissues. The compound lowered protein levels of phospho-p65 (Ser536), phospho-STAT3 (Tyr705), inducible nitric oxide synthases (iNOS), cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), IL-1ß and tumor necrosis factor α (TNF-α) in mouse swollen ears. In LPS-stimulated RAW264.7 cells, chrysoeriol also lowered levels of these proteins. In addition, chrysoeriol decreased the production of NO and prostaglandin E2; inhibited the phosphorylation of inhibitor of κB (Ser32), p65 (Ser536) and Janus kinase 2 (Tyr1007/1008); decreased nuclear localization of p50, p65 and STAT3; and down-regulated mRNA levels of pro-inflammatory cytokines IL-6, IL-1ß and TNF-α that are transcriptionally regulated by NF-κB and STAT3 in the cell model. CONCLUSION: We for the first time demonstrated that chrysoeriol ameliorates TPA-induced ear edema in mice, and that inhibition of JAK2/STAT3 and IκB/p65 NF-κB pathways are involved in the anti-inflammatory effects of chrysoeriol. This study provides chemical and pharmacological justifications for the use of chrysoeriol-containing herbs in treating inflammatory diseases, and provides pharmacological groundwork for developing chrysoeriol as a novel anti-inflammatory agent.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Toxidermias/tratamento farmacológico , Flavonas/farmacologia , NF-kappa B/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Toxidermias/metabolismo , Toxidermias/patologia , Regulação da Expressão Gênica , Proteínas I-kappa B/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos ICR , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7 , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/toxicidadeRESUMO
BACKGROUND: Receptor activator of NF-κB ligand (RANKL) facilitates differentiation of osteoclast precursors into osteoclasts, resulting in bone erosion in rheumatoid arthritis (RA) patients. Fibroblast-like synoviocytes (FLS) are the main cells for producing RANKL. Signal transducer and activator of transcription 3 (STAT3) signaling is activated in FLS of RA patients (RA-FLS), which has been linked to RANKL production. A two-herb formula (RL) comprising Rosae Multiflorae Fructus and Lonicerae Japonicae Flos is traditionally used for treating RA in China. We have found that a standardized ethanolic extract of RL (RLE for short) alleviates bone erosion in collagen-induced arthritis (CIA) rats. PURPOSE: This study aimed to determine whether RLE inhibits RANKL production and osteoclastogenesis in cell and rat models, and to explore the involvement of the STAT3 pathway in this inhibition. STUDY DESIGN AND METHODS: A CIA rat model, interleukin-6/soluble interleukin-6 receptor (IL-6/sIL-6R)-stimulated RA-FLS and a co-culture system (IL-6/sIL-6R-stimulated RA-FLS/peripheral blood mononuclear cells) were used to evaluate the effects of RLE. Micro-computed tomography analysis was used to observe bone erosion in CIA rats. Tartrate-resistant acid phosphatase staining was used to evaluate osteoclastogenesis. Western blotting and ELISA assays were employed to examine protein levels. RT-qPCR was used to detect mRNA levels. STAT3-over-activated RA-FLS were used to investigate the involvement of STAT3 signaling in the anti-osteoclastogenic effects of RLE. RESULTS: RLE alleviated bone erosion in joints of CIA rats. In both synovial tissues of CIA rats and IL-6/sIL-6R-stimulated RA-FLS, RLE downregulated the protein level of RANKL. In the co-culture system, RLE significantly and dose-dependently inhibited IL-6/sIL-6R-induced osteoclastogenesis. Mechanistic studies revealed that RLE lowered the protein level of phospho-STAT3 (Tyr705) in synovial tissues of CIA rats. In IL-6/sIL-6R-stimulated RA-FLS, RLE inhibited the activation/phosphorylation of a STAT3 upstream kinase Janus kinase 2 (Tyr1007/1008) and STAT3 (Tyr705), decreased the nuclear localization of STAT3, lowered mRNA levels of STAT3-transcriptionally regulated genes IL-1ß and TNF-α. RLE's inhibitory effects on RANKL production in RA-FLS gradually decreased when IL-6/sIL-6R doses increased. Over-activation of STAT3 diminished the inhibitory effects of RLE on RANKL production in IL-6/sIL-6R-stimulated RA-FLS, and attenuated the anti-osteoclastogenic effects of RLE in the co-culture system. CONCLUSION: We, for the first time, demonstrated that suppressing STAT3 signaling contributes to the inhibition of RANKL production and osteoclastogenesis, and thereby supports the mechanisms responsible for the reduction in bone erosion in RLE-treated CIA rats. This study provides further pharmacological groundwork for developing RLE as a modern anti-arthritic drug, and supports the notion that targeting STAT3 signaling is a viable strategy for managing bone erosion.
RESUMO
ETHNOPHARMACOLOGY RELEVANCE: Si-Jun-Zi-Tang (SJZT) is a traditional Chinese medicine formula used to treat chronic and debilitating diseases including melanoma. SJZT-based therapies have achieved good clinical outcomes in melanoma management. However, the pharmacological basis of SJZT for its clinical use in melanoma treatment is not fully understood. AIM OF THE STUDY: To investigate the anti-melanoma effects and mechanism of action of an ethanolic extract of SJZT. MATERIALS AND METHODS: SJZT was extracted using 50% ethanol. A murine B16 melanoma-bearing mouse model was employed to investigate the anti-melanoma effects of SJZT. microRNA (miRNA) and mRNA levels were examined by RT-qPCR, and protein levels were measured by Western blotting. RESULTS: SJZT significantly inhibited B16 tumor growth in mice. Mechanistic investigations revealed that SJZT elevated miR-34b (a tumor suppressing miRNA), and lowered c-Met (a miR-34b target gene) and ß-catenin (a downstream molecule of c-Met signaling) expression levels in the B16 tumors. CONCLUSIONS: In this study we found, for the first time, that SJZT exerts anti-melanoma effects and regulates the miR-34b/c-Met/ß-catenin pathway in a melanoma mouse model. Our findings provide pharmacological justifications for the clinical use of SJZT in treating melanoma.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Melanoma Experimental/tratamento farmacológico , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , beta Catenina/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Masculino , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-met/genética , Transdução de Sinais , Carga Tumoral/efeitos dos fármacosRESUMO
BACKGROUND: Metastasized melanoma is extremely difficult to treat. Activation of C-C chemokine receptor type 7 (CCR7) has been linked to melanoma metastasis. CCR7 can be directly regulated by miR-let-7. We have previously shown that an ethanolic extract of an herbal formula comprising Sophorae Flos and Lonicerae Japonicae Flos (SLE) inhibits melanoma cell migration and invasion. PURPOSE: In this study, we determined whether SLE suppresses melanoma metastasis, and whether regulation of miR-let-7a/f-CCR7 signaling is involved in the effect. STUDY DESIGN AND METHODS: Small RNA sequencing was conducted to compare miRNA expression profiles of B16F10 tumors dissected from SLE-treated or untreated mice. Western blot and RT-qPCR analyses were employed to examine protein and miRNA levels, respectively. A B16F10 melanoma lung metastasis mouse model was used to evaluate the effects of SLE on melanoma metastasis. MiR-let-7a/f-knockdown and CCR7-overexpression cell models were used to investigate the involvement of miR-let-7a/f-CCR7 signaling in the anti-metastatic effects of SLE. RESULTS: It was found that SLE upregulated levels of miR-let-7a/f in B16F10 melanoma tissues. SLE significantly elevated levels of miR-let-7a/f, lowered the protein level of CCR7, inhibited the phosphorylation of CCR7 downstream molecules p38 and JNK in B16F10 and A375 melanoma cells. SLE inhibited B16F10 melanoma lung metastasis in mice. SLE upregulated levels of miR-let-7a/f, and lowered protein levels of CCR7, MMP-2, MMP-9, phospho-p38 (Thr180/Tyr182) and phospho-JNK (Thr183/Tyr185) in melanoma-invaded lung tissues. Knockdown of miR-let-7a/f diminished the effects of SLE on CCR7 signaling in, and invasion of, melanoma cells. Overexpression of CCR7 lessened the effects of SLE in inhibiting the phosphorylation of p38 and JNK in, and the invasive capability of, melanoma cells. CONCLUSION: We for the first time demonstrated that SLE inhibits melanoma metastasis in mice, and that regulation of the miR-let-7a/f-CCR7 pathway contributes to the anti-metastatic mechanisms of SLE. These findings provide a pharmacological basis for developing SLE as a modern agent for treating metastatic melanoma. Additionally and importantly, this study suggests that regulating the miR-let-7a/f-CCR7 pathway is a novel strategy for controlling melanoma metastasis.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Melanoma Experimental/tratamento farmacológico , MicroRNAs/metabolismo , Extratos Vegetais/farmacologia , Receptores CCR7/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lonicera , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Masculino , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Extratos Vegetais/química , Receptores CCR7/genética , Sophora/químicaRESUMO
Colorectal cancer (CRC) is a leading cause of cancer-related morbidity and mortality worldwide. Signal transducer and activator of transcription 3 (STAT3) signaling is constantly activated in CRC, and has been proposed as a pathogenic factor and a therapeutic target of CRC. Ampelopsis Radix (AR), a traditional Chinese medicinal herb, possesses low toxicity and has long been used clinically for the treatment of cancers including CRC. Some constituents of AR have been reported to exert anti-cancer properties by targeting STAT3. However, the anti-CRC mode and mechanism of action of AR have not been fully elucidated. Here, we investigated the involvement of STAT3 signaling in the anti-CRC effects of AR. Results showed that AR reduced cell viability, induced cell apoptosis, and suppressed cell migration and invasion in human HCT-116 and SW480 CRC cells. Mechanistic studies showed that AR potently suppressed STAT3 and Src phosphorylation, and inhibited STAT3 nuclear localization in cultured CRC cells. AR also downregulated the expression of STAT3 target genes Mcl-1, Bcl-xL, and MMP-2 that are involved in cell survival and mobility. Moreover, the cytotoxic effect of AR was diminished by overexpressing STAT3C, a persistent active variant of STAT3. In conclusion, AR exerted anti-CRC effects in vitro and these effects are at least in part attributed to the inhibition of STAT3 signaling. Our findings provide a molecular justification for the traditional use of AR in treating CRC, and a pharmacological basis for developing AR-derived modern anti-CRC agent(s).